There is an emerging threat from multidrug-resistant Gram-negative bacterial pathogens, specifically, carbapenem-resistant Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas aeruginosa (e.g., ESKAPE pathogens). The resulting infections are often untreatable or treatable only with toxic antimicrobials. More troubling is the fact that the incidences of these infections are occurring with increasing frequency. Therefore, the CDC now categorizes such organisms in their top antibiotic resistance threat level. New anti-infective strategies are urgently needed. This multi-PI R01 application proposes a de novo medicinal chemistry design and de novo carbohydrate synthesis approach, which when coupled with functional characterization and cryo-EM enabled structure-guided design should lead to the rapid discovery of novel aminoglycoside (AG) antimicrobials with activity against resistant Gram-negative pathogens. The long-term expected outcomes are 1) the establishment of new synthetic methodology for systematic medicinal chemistry SAR-based exploration of the aminoglycoside chemical space and 2) the discovery of new aminoglycoside structural motifs with improved activity against resistant bacteria (e.g., AME, RMT- mediated resistance). The underlying hypothesis that guides our approach is the assumption that there are many carbohydrate structures that remain undiscovered due to the synthetic limitations of traditional carbohydrate and semi-synthetic approaches. In contrast, our total de novo synthetic approach enables the installation of a much broader range of carbohydrates in a stereochemically selective manner. Examples of structural variations that will be explored are aminoglycosides with rare aminosugar, linear sugar, and 2- deoxystreptamine (2-DOS) substitutions that are designed to evade known aminoglycoside resistance mechanisms.